We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. The use of label-free approaches for quantitative proteomics studies has been implemented. This methodology involves both data dependent (DDA) and data-independent analyses (Mse). The data obtained from depleted serum (depletion of the 14 most abundant proteins) followed by six different sample treatments has been acquired. The results obtained by data-independent analyses and by data-dependent analyses are being evaluated. Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions and thus travel more slowly than those ions that comprise a smaller cross-section. Thus, ion mobility serves as a useful means of orthogonal separation in unrelated molecules as well. This is a real benefit because the ability to identify and quantify proteins is directly linked to the power of chromatographic separations. 2. Fatty Acid Study. We performed a comparison of short chain fatty acids and amino acids in feces to determine if obesity has an effect on the metabolism of cells. It was shown previously that obesity can result in some epigenetic changes in colon epithelial cells and make them resemble tumor cells. 3. Buprenorphine Study. Buprenorphine is an opiate which is used to control pain in experimental animals by our CMB. Plasma concentrations of buprenorphine drop below therapeutic levels between 2 and 4 hours after subcutaneous injection. To achieve a longer therapeutic window, sustained release formulations and pluronic gel formulations are being investigated. Buprenorphine, norbuprenorphine, and their glucuronides in plasma have been quantified from initial formulations. 4. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 71 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. We are also developing an untargeted approach to analyze these molecules on another instrument. 5. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. Steroids are often present at low concentrations and exhibit low response in electrospray ionization. We are evaluating chemical derivatization procedures that will increase the signal intensity across the spectrum of hydroxy steroids and keto steroids. 6. BALF Studies. This project involves the pulmonary function of a knockout model of the Low Density Lipoprotein Receptor-related protein 1(LRP1). A novel pulmonary phenotype was identified and the disrupted mechanisms leading to this phenotype are being investigated. The receptor has over 30 ligands making the task of identifying changes in the concentration of any one ligand more difficult. We are using mass spectrometry to assay all of the relative concentrations of the ligands within the bronchoalveolar lavage fluid at one time, in addition to potential targets previously not considered. The data have been collected and the biostatisticians are doing statistics on the results. 7. NAD Studies. Per a request from the laboratory of Dr. Xiaoling Li, the MSRSG has recently developed an LC-MS-based panel for the relative quantitation of metabolites in the NAD pathway. Thus far, the method has been developed to include 11 compounds in the pathway and work is on-going to add 4 other molecules. This panel has been optimized thus far for the analyses of samples arising from cell culture, media, and tissue. In the future we plan to expand these analyses to serum and other biofluids. 8. Single Carbon Studies. Again in collaboration with the Li laboratory, a project that has been started recently in the MSRSG focuses on the relative quantitation of metabolites in the single carbon pathway. Thus far, the method has been developed to include 7 compounds in the pathway and work is on-going to add other molecules. This panel has been optimized thus far for the analyses of samples arising from cell culture, but future plans are to include tissues and biofluids. 9. Sulforaphane Study. Sulforaphane (SFN) is an anti-cancer compound that modulates inflammatory responses by suppressing the LPS-mediated expression of iNOS, COX-2, IL-1 and TNF-. Our observations also suggest activation of mitochondria function and related energy metabolism as an underlying pulmonary defense mechanism of SFN through Nrf2. Hyperoxia-induced acute lung injury (ALI) is a major lung disorder in adults affecting millions worldwide with 50-80% mortality rate. ALI is precipitated from various clinical disorders including pneumonia, sepsis, and major trauma, and is characterized by increased permeability and inflammation accompanying abnormal gas exchange in pulmonary airways and variable late phase responses. Nrf2 is a key transcriptional inducer of antioxidant and defense genes. As a susceptibility gene, we determined a protective role of Nrf2 in ALI-like phenotypes by use of gene knockout mice. Recent advances in redox research have provided the emerging recognition of the role of bioactive phytochemical antioxidants including SFN. We demonstrated that SFN administration significantly reduced ALI-like phenotypes caused by subsequent hyperoxia exposure in Nrf2-sufficient mice, but not in Nrf2-deficient mice. Differential lung transcriptome changes induced by SFN in Nrf2-sufficient and deficient mice suggested that SFN action through Nrf2 enhances pulmonary mitochondrial dynamics and metabolism to maintain the bioenergetic demands of lung cells against oxidative stress.